1. Field of the Invention
The present invention relates to methods for inhibiting cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF.alpha.) activities and treating disorders and diseases associated with elevated COX-2 and TNF.alpha. activities.
2. Description of the Background Art
The two isoforms of cyclooxygenase, cyclooxygenase 1 and 2 (COX-1 and COX-2), also referred to as prostaglandin endoperoxide synthase 1 and 2, are key enzymes in the conversion of arachidonic acid to prostaglandins, thromboxanes and other eicosanoids. It is believed that COX-1 and COX-2 have different physiologic functions due to striking differences in their tissue expression and regulation. COX-1 is a constitutive enzyme that is present at all times in the body and is responsible for the production of cytoprotective prostaglandins important for homeostatic functions, such as maintaining the integrity of the gastric mucosa, mediating normal platelet function, and regulating renal blood flow. In contrast, COX-2 is a rapidly inducible form of cyclooxygenase that leads to the production of proinflammatory prostaglandins. While COX-2 expression is highly restricted under basal conditions, it is dramatically up-regulated during inflammation.
Prostaglandins are produced at elevated levels in inflamed tissues including rheumatoid synovium. The prostaglandins PGE.sub.1 and PGE.sub.2 contribute to synovial inflammation by increasing local blood flow and potentiating the effects of mediators, such as bradykinin and IL-1.beta. that induce vasopermeability. PGE.sub.2 has also been shown to trigger osteoclastic bone resorption, suggesting that this molecule may contribute to the pathophysiology of joint erosion in rheumatoid arthritis. The involvement of COX-2 and the elevated production of prostaglandins are associated with a variety of diseases and disorders, such as brain ischemia and cancers, as well as diseases and disorders in which elevated levels of nitric oxide is present.
Studies have indicated that nitric oxide (NO), a recently recognized multi-functional mediator produced by and acting on various cells, modulates the activity of COX-2 (Salvemini et al, 1993) and participates in inflammatory and autoimmune-mediated tissue destruction. The effect of NO on COX-2 is dose-dependent. Low levels of NO activate COX-2. In contrast, large amounts of NO produced by inducible nitric oxide synthase (iNOS) can inhibit the induction of COX-2 and suppress the formulation of COX-2 metabolites. Salvemini and co-workers recently demonstrated inhibition of inflammatory response by modulation of NO production in various animal models of inflammation. Nitric oxide formation has been found to be increased in autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease), and several classic inflammatory symptoms (erythema, vascular leakiness) are reversed by NOS inhibitors. However, while NOS inhibitors, such as N.sup.G -monomethyl-L-arginine acetate (L-NMMA), inhibit the production of NO, they also appear to have the property of augmenting PGE.sub.2 production due to attenuation of NO-induced down-regulation of PGE.sub.2 production. Thus, most NOS inhibitors up-regulate COX-2 and the production of PGE.sub.2 in a NO-dependent manner.
Tumor necrosis factor alpha (TNF.alpha.), a pleiotropic cytokine, produces a broad scope of injurious effects which also makes it an important target for therapeutic intervention. TNF.alpha. is involved in the pathophysiology of arthritis, AIDS, cancer, autoimmune diseases (immune complex diseases), lung fibrosis, multiple sclerosis, skin DTL reactions, and bacterial and parasitic infections. The gene for human TNF.alpha. encodes a prohormone that is inserted into the cell membrane as a polypeptide with a MW of 26 kD. This membrane-bound form of TNF.alpha. is bioactive as assayed by cell cytotoxicity and has been implicated in the paracrine activities of TNF.alpha. in tissues. In response to lipopolysaccharide (LPS) and other stimuli, the 26 kD form of proTNF.alpha. is proteolytically cleaved (by a metalloprotease referred to as TNF.alpha. convertase) into a soluble 17 kD polypeptide. TNF.alpha. binds to its cognate receptors (p55 and p75) as a bioactive trimer, and signals within the cell.
Systemic exposure to soluble recombinant TNF.alpha. (in quantities that could be produced endogenously by the host during infection) causes an acute syndrome of shock and tissue injury that is virtually indistinguishable from septic shock syndrome. This effect is followed by capillary leakage syndrome, hypoxia, pulmonary edema, and multiple organ failure. Such observations highlight the importance of TNF.alpha. as an important therapeutic target for various pathophysiological conditions.
Studies in animal models of arthritis and human rheumatoid arthritis indicate that TNF.alpha. may be a pivotal cytokine involved in these disease processes. Injection of anti-TNF.alpha. antibodies and/or soluble TNF.alpha. receptor has proven to be highly effective in reducing clinical signs and symptoms in controlled studies. Extension of these studies in phase II and III clinical trials (in rheumatoid arthritis) has yielded very encouraging results, again indicating that neutralizing the effects of TNF.alpha. may have profound effects on disease progression in various inflammatory diseases, including arthritis.
The matrix metalloproteases (MMPs) have long been recognized to play an important role in cartilage degradation in both rheumatoid arthritis (RA) and osteoarthritis (OA). The synovium and cartilage are the most important sources of degradative enzymes that contribute to the pathophysiology of OA and RA. These enzymes include metalloproteases, serine proteases, proteoglycanases and thiol proteases. The two main families of MMPs believed to be responsible for cartilage degradation are collagenases and proteoglycanases.
Early studies by three independent groups have demonstrated that broad-spectrum inhibitors of matrix metalloproteases can specifically inhibit the release of membrane proTNF.alpha. (but not IL-1.beta. or IL-6) from various cell surfaces, including rheumatoid arthritis synovial cell cultures. This inhibitor of proTNF.alpha. processing could protect mice against a lethal dose of administered endotoxin. The "TNF.alpha. convertase" activity was isolated using these inhibitors as ligands by affinity purification which resulted in identification of an 80 kD protein with the capacity to cleave the Gln-Ala-.dwnarw.-Val-Arg (SEQ ID NO:1) sequence of proTNF.alpha..
Doxycycline and minocycline are members of the tetracycline family of broad-spectrum antibiotics. During recent years, it has been established that tetracyclines, which are rapidly absorbed and have a prolonged half-life, exert biological effects independent of their antimicrobial activity (Golub et al, 1991; Golub et al, 1992; Uitto et al, 1994). Such effects include inhibition of matrix metalloproteases, including collagenase (MMP-1), gelatinase (MMP-2) and stromelysin (MMP-3) activity, and prevention of pathogenic tissue destruction (Golub et al, 1991). In inflammatory arthritides such as rheumatoid arthritis, these matrix metalloproteases have been identified in homogenates and cultures of rheumatoid synovium, detected in inflammatory synovial fluids and localized immunologically and by in situ hybridization in proliferative pannus and synovium (Brinckerhoff, 1991). These metalloproteases are known to be up-regulated in OA-affected joints (Greenwald, 1994; Mohtai et al, 1993). Interestingly, Yu et al (1992) have also shown that prophylactic administration of doxycycline markedly reduced the severity of OA in dog models. To assess the safety and efficacy of minocycline (a semisynthetic tetracycline) in the treatment of arthritis, a double-blind, randomized, multicenter trial indicated that the drug was safe and effective for patients with mild and moderate arthritis (Tilley et al, 1995). Furthermore, recent studies have also suggested that tetracyclines and inhibitors of metalloproteases inhibit tumor progression (DeClerck et al, 1994), bone resorption (Rifkin et al, 1994) and angiogenesis (Maragoudakis et al, 1994), and may have anti-inflammatory properties (Ramamurthy et al, 1994).
The laboratory of the present inventors recently observed that tetracycline and chemically modified tetracyclines inhibit nitric oxide production in macrophages and OA-affected cartilage where NOS expression is inhibited at the level of post transcriptional modification of NOS (Amin et al, 1995b, 1997b). The laboratory of the present inventors have also recently observed that human arthritis-affected cartilage in ex vivo conditions (in the absence or presence of cytokines+endotoxins) releases substantial amount of NO and PGE.sub.2, and the specific inhibition of NO production augments PGE.sub.2 production in a NO-dependent fashion (Amin et al, 1997a).
Tetracyclines have also been found to be effective for treating collagenase activity in periodontal disease and diabetes (U.S. Pat. No. 4,666,897 and No. 4,704,383), for inhibiting excessive phospholipase A.sub.2 -activity (U.S. Pat. No. 5,523,297), and for inhibiting elastase activity (U.S. Pat. No. 5,773,430)
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